nedd4-mediated increase in hiv-1 gag and env … immunity following dna-vaccination of balb ......

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Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice Brad Lewis, Stephen Whitney, Lauren Hudacik, Lindsey Galmin, Maria Cecilia Huaman, Anthony D. Cristillo* Advanced BioScience Laboratories, Inc., Rockville, Maryland, United States of America Abstract The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIV SF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4 + and CD8 + T cells following SHIV SF162P3 -infection of naı ¨ve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca 2+ / calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNc but had a trend of increased Gag-specific IL-6, IL-17A and TNFa that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation. Citation: Lewis B, Whitney S, Hudacik L, Galmin L, Huaman MC, et al. (2014) Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice. PLoS ONE 9(3): e91267. doi:10.1371/journal.pone.0091267 Editor: Clive M. Gray, University of Cape Town, South Africa Received October 16, 2013; Accepted February 10, 2014; Published March 10, 2014 Copyright: ß 2014 Lewis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: All authors are affiliated with Advanced BioScience Laboratories, Inc., as listed in the Competing Interests section of the online manuscript form. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: [email protected] Introduction The role of ubiquitin ligases on late stage viral processing and budding has been investigated by many laboratories working on different viruses including but not limited to human immunode- ficiency virus-1 (HIV-1), feline immunodeficiency virus (FIV), Rous sarcoma virus (RSV), Ebola virus, Avian sarcoma virus and Murine leukemia virus (MLV) [1–5]. Structural proteins, such as HIV-1 Gag, have been found to be mono-ubiquitinated and depletion of intracellular-free ubiquitin pools, using proteasome inhibitors, shown to inhibit budding [6–8]. While many studies have been conducted in order to dissect the mechanism(s) by which this ubiquitination occurs and is regulated, the signaling pathways in host target cells that influence viral budding are not completely understood. While most regions of HIV-1 Gag and other retroviral structural proteins appear to be dispensable for budding, an essential region has been identified as the late assembly (L-) domain [9–13]. The core element in the L-domain has been shown to include the following conserved sequences: PTAP, PPxY, LxxLF, and YPxL [10–12,14,15]. The core element has been found to be required for efficient pinching off of the virus bud [9,11,12]. Furthermore, expression of HIV-1, RSV or BLV Gag L-domain deletion mutants resulted in a budding defect in which host cells remained covered with viral particles tethered to the membrane [7,8]. The L-domain core sequences (PTAP, PPxY, LxxLF and YPxL) are well conserved throughout the retroviridae family and thus support their importance in viral budding and pathogenesis. Ongoing studies seeking to bridge the gap ‘‘mechanistically’’ between viral budding, ubiquitination and the L-domain core element have identified class E vacuolar protein sorting factors, tumor susceptibility gene product (Tsg101) [16–18] and AIP1/ ALIX [1], as key mediators of HIV-1 Gag trafficking and viral budding. Tsg101, an ESCRT-I (endosomal sorting complex required for transport I) component, has been shown to interact with the PTAP motif of HIV-1 Gag p6 [19,20] and mediate budding via multivesicular bodies [21,22]. Studies have clearly demonstrated, using a dominant negative Tsg101 [23–27] or Tsg101-targeted siRNA [16], that this ESCRT-I component is critical for HIV-1 budding. AIP1/ALIX has also been shown to play a key role by binding to the YPxL motif of HIV-1 Gag and thereby associating HIV-1 Gag and Tsg101 to the endosomal complex ESCRT-III. Conversely, it has been shown that viruses of the ‘‘PPxY’’ L-domain type such as HTLV-I, RSV and Ebola PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e91267

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Page 1: Nedd4-Mediated Increase in HIV-1 Gag and Env … Immunity following DNA-Vaccination of BALB ... vitro and its potential as an immune modulator in HIV ... 1 (HIV-1), feline immunodeficiency

Nedd4-Mediated Increase in HIV-1 Gag and Env Proteinsand Immunity following DNA-Vaccination of BALB/c MiceBrad Lewis, Stephen Whitney, Lauren Hudacik, Lindsey Galmin, Maria Cecilia Huaman,

Anthony D. Cristillo*

Advanced BioScience Laboratories, Inc., Rockville, Maryland, United States of America

Abstract

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins,are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteinswith intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 buddinghave been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function forNedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 followingSHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immunemodulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cellsfollowing SHIVSF162P3-infection of naı̈ve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfectionof Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gagor Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularlywith plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 butnot anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNc but had a trend ofincreased Gag-specific IL-6, IL-17A and TNFa that was not seen following Env stimulation. Based on our initial findings,Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants furtherinvestigation.

Citation: Lewis B, Whitney S, Hudacik L, Galmin L, Huaman MC, et al. (2014) Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity followingDNA-Vaccination of BALB/c Mice. PLoS ONE 9(3): e91267. doi:10.1371/journal.pone.0091267

Editor: Clive M. Gray, University of Cape Town, South Africa

Received October 16, 2013; Accepted February 10, 2014; Published March 10, 2014

Copyright: � 2014 Lewis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The authors have no support or funding to report.

Competing Interests: All authors are affiliated with Advanced BioScience Laboratories, Inc., as listed in the Competing Interests section of the onlinemanuscript form. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

* E-mail: [email protected]

Introduction

The role of ubiquitin ligases on late stage viral processing and

budding has been investigated by many laboratories working on

different viruses including but not limited to human immunode-

ficiency virus-1 (HIV-1), feline immunodeficiency virus (FIV),

Rous sarcoma virus (RSV), Ebola virus, Avian sarcoma virus and

Murine leukemia virus (MLV) [1–5]. Structural proteins, such as

HIV-1 Gag, have been found to be mono-ubiquitinated and

depletion of intracellular-free ubiquitin pools, using proteasome

inhibitors, shown to inhibit budding [6–8]. While many studies

have been conducted in order to dissect the mechanism(s) by

which this ubiquitination occurs and is regulated, the signaling

pathways in host target cells that influence viral budding are not

completely understood.

While most regions of HIV-1 Gag and other retroviral

structural proteins appear to be dispensable for budding, an

essential region has been identified as the late assembly (L-)

domain [9–13]. The core element in the L-domain has been

shown to include the following conserved sequences: PTAP, PPxY,

LxxLF, and YPxL [10–12,14,15]. The core element has been

found to be required for efficient pinching off of the virus bud

[9,11,12]. Furthermore, expression of HIV-1, RSV or BLV Gag

L-domain deletion mutants resulted in a budding defect in which

host cells remained covered with viral particles tethered to the

membrane [7,8]. The L-domain core sequences (PTAP, PPxY,

LxxLF and YPxL) are well conserved throughout the retroviridae

family and thus support their importance in viral budding and

pathogenesis.

Ongoing studies seeking to bridge the gap ‘‘mechanistically’’

between viral budding, ubiquitination and the L-domain core

element have identified class E vacuolar protein sorting factors,

tumor susceptibility gene product (Tsg101) [16–18] and AIP1/

ALIX [1], as key mediators of HIV-1 Gag trafficking and viral

budding. Tsg101, an ESCRT-I (endosomal sorting complex

required for transport I) component, has been shown to interact

with the PTAP motif of HIV-1 Gag p6 [19,20] and mediate

budding via multivesicular bodies [21,22]. Studies have clearly

demonstrated, using a dominant negative Tsg101 [23–27] or

Tsg101-targeted siRNA [16], that this ESCRT-I component is

critical for HIV-1 budding. AIP1/ALIX has also been shown to

play a key role by binding to the YPxL motif of HIV-1 Gag and

thereby associating HIV-1 Gag and Tsg101 to the endosomal

complex ESCRT-III. Conversely, it has been shown that viruses of

the ‘‘PPxY’’ L-domain type such as HTLV-I, RSV and Ebola

PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e91267

Page 2: Nedd4-Mediated Increase in HIV-1 Gag and Env … Immunity following DNA-Vaccination of BALB ... vitro and its potential as an immune modulator in HIV ... 1 (HIV-1), feline immunodeficiency

virus utilize Nedd4 (E3 ubiquitin ligase) family members to

mediate viral trafficking and budding [17,28–36].

While the role of Nedd4 on viral egress for ‘‘PPxY’’ type viruses

has been well studied, several laboratories in recent years have

elucidated a role for Nedd4 and Nedd4 family members on viruses

containing PTAP and YPxL motifs. To this end, the Nedd4-like

(Nedd4L) protein was shown to rescue HIV-1 budding defects

caused by a lack of Tsg101- and ALIX late domains [5]. Nedd4

has also been shown to be recruited by ALIX in facilitating HIV-1

budding via ALIX-dependent ubiquitination [37]. Furthermore,

exogenous expression of Nedd4-2 s was found to increase HIV-1

budding that was shown to be dependent on ubiquitination [38]

and the truncated C2 domain [39].

We here identify increased Nedd4 protein levels in both CD4+

and CD8+ T cells following SHIVSF162P3-infection of naı̈ve

macaques. Transient co-transfection of 293 cells with HIV-

1HXB2 and Nedd4 subsequently demonstrated a Nedd4-mediated

increase in p24 and gp120 levels that was dependent on the Ca2+/

calmodulin-regulated phospholipid binding C2 domain but not

ubiquitin ligase activity. Vaccination of BALB/c mice with Nedd4

co-administration yielded increase serum anti-p24 but not anti-

gp120 antibodies. Nedd4 co-injection was found to have no affect

on Gag- or Env-specific IFNc but had a trend of increased Gag-

specific IL-6, IL-17A and TNFa that was not seen following Env

stimulation.

Materials and Methods

Housing and Care of Rhesus MacaquesThe animals in this study were Indian rhesus macaques (Macaca

mulatta) and were housed at the Advanced BioScience Laborato-

ries, Inc. (ABL) animal facility. All animals were cared for and

procedures performed under a protocol approved by the ABL

Animal Care and Use Committee (animal welfare assurance no.

A3467-01; protocol no. AUP308). Furthermore, the macaques in

this study were managed according to the animal husbandry

program of the ABL Animal Facility, which aims at providing

consistent and excellent care to nonhuman primates at the

vivarium. This program operates based on the laws, regulations,

and guidelines promulgated by the United States Department of

Agriculture (e.g., the Animal Welfare Act and its regulations, and

the Animal Care Policy Manual), Institute for Laboratory Animal

Research (e.g., Guide for the Care and Use of Laboratory

Animals, 8th edition), Public Health Service, National Research

Council, Centers for Disease Control, and the Association for

Assessment and Accreditation of Laboratory Animal Care

(AAALAC) International.

The nutritional plan utilized by the ABL Animal Facility

consisted of twice daily feeding of Labdiet 5045 High Protein

Primate Diet and food intake was closely monitored by Animal

Research Technicians. This diet was also supplemented with a

variety of fruits, vegetables, and other edible objects as part of the

environmental enrichment program established by the Veterinary

staff and enrichment Technician. Pairing of animals as part of the

environmental enrichment program was managed by the enrich-

ment technician. All primary enclosures and animal rooms were

cleaned daily with water and sanitized at least once every two

weeks. Viral challenges were performed under anesthesia (Keta-

mine administered at 10 mg/kg) and all efforts were made to

minimize suffering. None of the animals were euthanized as part

of this study.

ABL’s routine health surveillance consists of physical examina-

tions, tuberculin (TB) tests as well as clinical tests and observations.

Animals are periodically screened for viruses and pathogens such

as STLV, SRV, measles and herpes B virus, and SIV using

serological and sensitive real time PCR-based assays developed at

ABL. Animal observations and general health checks will be

performed twice daily, seven days a week, on all animals assigned

to this program. Daily observations of all animals will be done to

assess their health and well being. Animals are observed for

changes in stool condition, food consumption, evidence of trauma,

signs of pain or distress, and the appearance of any clinical signs

that indicate ill health. ABL has an established environmental

enrichment plan for nonhuman primates compliant with the

Animal Welfare Act. This enrichment plan includes social

interaction through group housing, sensory and cognitive enrich-

ment, and identification of and individualized treatment for

psychological distress.

The program veterinarian is authorized to make decisions as to

whether animals meet criteria that constitute humane endpoints

that will result in removal of animals from study. The program

veterinarian is in close communication with the principal

investigator regarding the health status and medical conditions

of animals enrolled in studies. SHIV-infected animals were used

for this study and SHIV-infected animals may develop AIDS like

syndromes, such as market drop in blood CD4+ T cells, severe

weight loss, diarrhea and may acquire opportunistic infections.

Under such circumstances these animals may be euthanized

following the veterinarian’s recommendation.

Virus Challenge of Rhesus MacaquesFour male, juvenile, naı̈ve Indian origin rhesus macaques (L861,

L866, L867, L868) were challenged intra-rectally with a single,

high dose (TCID in rhesus PBMC: 1.026104/ml) concentration of

SHIVSF162P3.

Plasma ViremiaAnimals were bled periodically following challenge and plasma

viral load was assessed using a sensitive real time nucleic acid

sequence-based amplification assay (NASBA) to quantitate SIV

RNA [40].

CD4 T CELL ImmunophenotypingFollowing challenge of rhesus macaques, animals were bled

periodically and absolute CD4 T cell measurements were

performed using the BD Biosciences TruCountTM platform using

an immunophenotyping panel consisting of anti-CD3/anti-CD4/

anti-CD8/anti-CD45 antibodies (BD Biosciences, San Diego,

CA).

Intracellular Detection of Nedd4 by Flow CytometryIntracellular levels of Nedd4 were measured by cytometry in

rhesus macaque PBMC. Briefly, cells were pelleted by centrifu-

gation at 11006g for 5 min and were then resuspended in 100 ml

FACS wash buffer (BD Biosciences) and stained with a cocktail of

fluorochrome conjugated anti-CD3, anti-CD4 and anti-CD8

antibodies (BD Biosciences) at room temperature for 30 min.

Cells were then washed with FACS wash buffer and resuspended

in 250 ml Cytofix/Cytoperm buffer (BD Biosciences) for 15 min at

4uC. Following incubation, 2 ml of Perm/Wash buffer (BD

Biosciences) was added to cells. Cells were pelleted by centrifu-

gation and then resuspended in Perm/Wash buffer containing a

rabbit anti-Nedd4 antibody (1:100; Upstate, Charlottesville, VA).

Cells were incubated for 15 min at 4uC, washed with 2 ml of

Perm/Wash buffer followed by centrifugation and staining with a

secondary goat anti-rabbit-RPE antibody (1:50; Southern Biotech,

Birmingham, AL) for 15 min at 4uC. Cells were washed with 2 ml

Nedd4 Increases HIV Immunity Post Vaccination

PLOS ONE | www.plosone.org 2 March 2014 | Volume 9 | Issue 3 | e91267

Page 3: Nedd4-Mediated Increase in HIV-1 Gag and Env … Immunity following DNA-Vaccination of BALB ... vitro and its potential as an immune modulator in HIV ... 1 (HIV-1), feline immunodeficiency

of Perm/Wash buffer, centrifuged and then resuspended in 200 ml

of FACS wash buffer (BD Biosciences). Acquisition of cells was

performed by cytometry using a FACScaliburTM (BD Biosciences).

Constructs and Molecular ClonesThe HIV-1 molecular clone, HXB2, was a generous gift from

Dr. Marvin Reitz (Institute of Human Virology, University of

Maryland Biotechnology Institute). Human Nedd4 and Nedd4-2

plasmid DNA were generously provided by Dr. Hughes Abriel

(Institute of Pharmacology and Toxicology, University of Lau-

sanne, Lausanne, Switzerland) and rat Nedd4 (rNedd4) and

Nedd4CSmut (rNedd4Csmut) plasmid DNA were gifts from Dr.

Daniela Rotin (University of Toronto, Toronto, Canada). The

hNedd4C2mut plasmid was generated by digestion of the hNedd4

construct with PpMuI and NdeI restriction enzymes (New

England Biolabs, Ipswich, MA). Following isolation of the

1134 bp digested fragment, using the QIAquick Gel Extraction

kit (Qiagen, Valencia, CA), PCR was performed using the

following primers: Nedd4C2mut-F:59GTACATCAAGTGTAT-

CATATGCCAAGTACGCCCCCTAT-39; Nedd4C2mut-R:59-

ATAGGTCCTTCCAAGGAGCCCGAACACCTCCACCGC-

39. The resulting PCR product (549 bp) was digested with PpMuI

and NdeI restriction enzymes and subcloned into the PpMuI/

NdeI-digested hNedd4 plasmid. Successful subcloning was verified

by restriction digestion and sequencing. Corresponding empty

plasmids for hNedd4, hNedd4-2, rNedd4 and rNedd4CSmut were

used as controls in addition to the empty pEGFP construct.

Transient Transfections293 cells (ATCC, Manassas, VA) were transiently transfected

using Lipofectamine 2000 (Life Technologies, Carlsbad, CA)

according to the manufacturer’s recommended protocol. Briefly,

293 cells (66106) were suspended in 12 ml of RPMI-1640 (Quality

Biological, Gaithersburg, MD), supplemented with 10% heat-

inactivated fetal calf serum (FCS) (HyClone, Logan, Utah), 2 mM

L-glutamine (Quality Biological), and 50 mM 2-mercaptoethanol

(Sigma, St. Louis, MO), termed antibiotic-free RPMI-1640. Cells

were then transferred to a 100 mm tissue culture dish and

incubated for 24 hr at 37uC with 5% CO2. On the day of

transfection, 60 ml of Lipofectamine 2000 was diluted with Opti-

Mem I Reduced Serum Medium (Life Technologies) to give a final

volume of 1.5 ml and incubated for 5 min at room temperature. A

total of 24 mg DNA (i.e., 12 mg of HXB2 plus 12 mg of hNedd4

plasmid) was diluted in Opti-Mem I Reduced Serum Medium to

give a final volume of 1.5 ml. The diluted DNA mixture was

added to the Lipofectamine mixture and incubated at room

temperature for 20 min. The DNA/Lipofectamine mixture (3 ml)

was then added to the 100 mm culture of 293 cells and incubated

for 24 hr at 37uC with 5% CO2. After 24 hr, the medium was

replaced with 15 ml of RPMI 1640 supplemented with 10% heat-

inactivated FCS, 2 mM L-glutamine, 50 mM 2-mercaptoethanol,

100 U/ml penicillin (MediaTech, Herndon, VA) and 100 mg/ml

streptomycin (MediaTech), termed 10% cRPMI-1640. At 24, 48

or 72 hr, cells and cell supernatants were collected by centrifu-

gation at 11006g for 5 min. For siRNA transfection studies, 293

cells (0.86106 cells) were transiently transfected in 6-well tissue

culture dishes, using Lipofectamine 2000 as outlined above, with

HXB2 (0.75 mg) and siRNA (4 mg; Qiagen) generated against:

Nedd4 (59-TAGAGCCTGGCTGGGTTGTTTTG-39) or con-

trol GFP (59-GCACAAGCTGGAGTACAACTACA-39).

HIV-1 p24 and gp120 Antigen Capture AssaysHIV-1 p24 and gp120 antigen capture ELISAs (Advanced

BioScience Laboratories, Kensington, MD) were used to quantify

supernatant and intracellular HIV-1 p24 and gp120, respectively,

as recommended by the manufacturer and previously described

[41].

Radioimmunoprecipitation Assay (RIPA)At 32 hr post-transfection, HXB2-transfected 293 cells with or

without hNedd4 co-transfection, were labeled overnight with 35S-

Met (250 mCi) in Methionine free DMEM (without L-glutamine,

L-Met, L-Cys; Invitrogen) supplemented with 2% FBS (heat-

inactivated and dialyzed), 2 mM L-glutamine (Quality Biological),

1 mM sodium pyruvate (Quality Biological) and 4 mM L-Cys

(Sigma, St. Louis, MO) at 37uC with 5% CO2. After labeling,

media was centrifuged at 11006g for 5 min at room temperature.

The clarified media was added to PBS-TD lysis buffer (final

concentration: 1X PBS, 0.5% Triton X-100 (Biorad), 1%

Deoxycholic acid (Sigma), supplemented with 1 ml/ml Protease

Inhibitor cocktail (Sigma; containing pepstatinA, E-64, bestatin,

leupeptin, aprotinin and 4-(2-aminoethyl) benzenesulfonyl fluo-

ride) followed by incubation at room temperature for 30 min.

Cells were lysed in PBS-TD lysis buffer supplemented with

Protease Inhibitor cocktail (1 ml/ml) and benzonase (1 ml/ml

Novagen; Madison, WI) and incubated for 30 min at room

temperature. Cell lysates and media were precleared by incubation

with normal human serum and protein-A sepharose (PAS; GE

Healthcare). HIV-1 proteins were immunoprecipitated from

precleared cell lysates and mediae with PAS and anti-HIV(+)

human serum (Sera Care Life Sciences Inc., Oceanside, CA).

Uninfected (HIV-1(2)) normal human serum was used as a

control (data not shown). After a 2 hr immunoprecipitation at

room temperature, samples were washed with PBS-TD buffer,

resuspended in 1X Leammli sample buffer, boiled for 2 min and

centrifuged at 11006g for 1 min to clarify samples. Proteins were

resolved by 10% SDS-PAGE. Gels were incubated for 45 min in

30% methanol and 7% acetic acid and incubated for 30 min in

Amplify buffer (GE Healthcare). Gels were dried to filter paper,

under vacuum, for 2 hr at 80uC and exposed to film for 24 hr at –

70uC.

Immunoprecipitations/Western Blot Analysis293 cells were transiently transfected, as described above, with

HXB2 alone or in combination with hNedd4, hNedd4C2mut,

hNedd4-2, rNedd4 or rNedd4Csmut plasmid DNA. Following

transfection (48 hr), cells were harvested by centrifugation at

11006g for 5 min at 4uC, washed once with 1X PBS and

resuspended at a concentration of 26106 cells/ml in lysis buffer A

[(1% NP-40 (Sigma), 0.15 M NaCl, 25 mM Tris pH 7.5, 1 mM

EDTA, 1 mM PMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin,

1 mM sodium orthovanadate (Sigma)]. Cells were incubated on ice

for 20 min and then centrifuged at 17,5306g for 10 min at 4uC.

The detergent soluble fraction (30 ml of supernatant) was mixed

with an equal volume (30 ml) of 2X Laemmli sample buffer

whereas the detergent insoluble fraction was resuspended in 1X

Laemmli sample buffer. Samples were heated for 5 min at 95uCand proteins were separated by 10% SDS-PAGE (Protogel,

National Diagnostics, Atlanta, GA). The remaining detergent

soluble fractions (cell supernatants) were incubated with either a

rabbit anti-GFP polyclonal antibody (Clontech, Mountain View,

CA) or anti-Nedd4 monoclonal antibody (BD Biosciences) for 2 hr

at 4uC, after which, protein A agarose (Santa Cruz Biotechnol-

ogies) was added and samples were incubated for an additional

30 min at 4uC. Samples were washed three times in buffer B (1%

NP-40, 0.15 M NaCl, 25 mM Tris pH 7.5, 1 mM EDTA, 1 mM

sodium orthovanadate), resuspended in 1X Laemmli sample

buffer and heated for 5 min at 95uC. Proteins were separated by

Nedd4 Increases HIV Immunity Post Vaccination

PLOS ONE | www.plosone.org 3 March 2014 | Volume 9 | Issue 3 | e91267

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10% SDS-PAGE and transferred to polyvinylidene difluoride

(PVDF) membranes (Millipore, Bedford, MA), immunoblotted

with either an anti-GFP monoclonal antibody (Clontech), rabbit

anti-mouse Nedd4 polyclonal antibody (BD Biosciences) and

detected by enhanced chemiluminesence (ECL) (GE Healthcare)

according to the manufacturer’s instructions.

Transient Transfection and Luciferase Report AssaysTransient transfection of Jurkat T cells was performed by

electroporation using the Gene Pulser (Biorad, Hercules, CA)

according to the manufacturer’s instructions. The LTR-luc Firefly

luciferase reporter construct was co-transfected with the hNedd4

construct and a reporter vector that contains a cDNA encoding

Renilla luciferase (pRL-TK) under the control of the herpes

simplex virus thymidine kinase promoter (Promega, Madison, WI).

pRL-TK was used to control for transfection efficiency. Jurkat T

cells (107 cells) were transfected with 10 mg of LTR-luc, 15 mg of

hNedd4 and 0.25 mg of pRL-TK by electroporation (270V and

960 mF). Cells were incubated for 24 hr at 37uC, 5% CO2 in air

and then stimulated with phorbol 12-myristate-13-acetate (PMA;

Calbiochem, La Jolla, CA) and Ionomycin (Iono; Calbiochem, La

Jolla, CA) as indicated. The Dual Luciferase assay (Promega,

Madison, WI) was performed to determine both Firefly and

Renilla luciferase activities in cell lysates. Briefly, stimulated cell

suspensions were transferred to Eppendorf tubes and pelleted by

centrifugation at 5006g for 5 min. Cell pellets were washed once

with 1X PBS and then lysed with 50 ml of 1X Promega passive

lysis buffer. Samples were vortexed for 30 sec, incubated at room

temperature for 15 min and pelleted again for 5 min at 20,0006g.

The luminescence of 100 ml of luciferase assay reagent added to

20 ml of each lysate was recorded using a Centro LB960

luminometer (EG&G Berthold, Gaithersburg, MD). Finally,

100 ml of Stop & GloTM reagent was added to the sample and

a second luminescence reading recorded (Renilla luciferase).

AntigensThe codon optimized gene coding for HIV-1Ba-L gp120 were

generous gifts from Dr. Marvin Reitz (Institute of Human

Virology, University of Maryland Biotechnology Institute). Mosaic

HIV-1 Gag immunogens (Clades A, B and C) were designed as

described [42,43] using the Mosaic Vaccine Designer software

(http://www.hiv.lanl.gov/content/sequence/MOSAIC/

makeVaccine.html). The selected mosaic gag sequences were based

on both optimal 9-mer coverage and the breadth of predicted

epitope responses as described [44] using the BIMAS software tool

(http://www-bimas.cit.nih.gov/cgi-bin/molbio/

ken_parker_comboform). Plasmid DNA used for mice immuniza-

tions, encoding clade A, B and C gag sequences, was under the

control of a CMV promoter. Plasmid DNA expressing HIV-1Ba-L

envelope was used for immunization of mice as previously

described [45].

Mice ImmunizationsBALB/c mice (n = 5 per group; 5–6 week old females; Taconic,

Hudson, New York) were immunized at weeks 0, 2 and 4 by

intramuscular injection with plasmid DNA encoding human

Nedd4 (100 mg) alone (Group 1), HIV-1 gag (pool of 100 mg each

of Mosaic Clade A, B, C)+HIV-1Ba-L env (100 mg) (Group 2) or

human Nedd4 (100 mg)+HIV-1 gag (pool of 100 mg each of Mosaic

Clade A, B, C)+HIV-1Ba-L env (100 mg) (Group 3). Group 4 mice

(n = 3) were left un-immunized and served as naı̈ve controls. Two

weeks following the final immunization (week 6), mice were

sacrificed, splenocytes and serum collected and serum antibody

responses and splenocyte T-cell responses were performed.

Typically, the immunological responses are minimal and are

often undetectable in naı̈ve, control animals. All animal studies

were reviewed and approved by the Institutional Animal Care and

Use Committee (IACUC) at Advanced BioScience Laboratories.

PeptidesGag peptides, used for ex vivo stimulation of splenocytes, were

obtained through the NIH AIDS Reagent Program, Division of

AIDS, NIAID, NIH: HIV-1 Consensus A Gag (15-mer) Peptides -

Complete Set; HIV-1 Consensus B Gag (15-mer) Peptides -

Complete Set; HIV-1 Consensus C Gag (15-mer) Peptides -

Complete Set. For each clade, Gag peptides were resuspended in

one peptide pool and used for ex vivo stimulation at a final per

peptide concentration of 1 mg/ml. For Clade B Env (HIV-1Ba-L),

79 peptides (15-mers) with 11 amino acid overlapping residues

were synthesized that comprise the gp120 Env protein sequence.

Clade B Env (HIV-1Ba-L) peptides were resuspended in one

peptide pool and used for stimulation at a final per peptide

concentration of 1 mg/ml.

Binding Antibody AssaySerum samples were tested for Env-specific antibodies and Gag-

specific antibodies using an enzyme-linked immunosorbent assay

(ELISA) as previously described [45]. Antibodies were detected

against HIV-1 p24 Gag or HIV-1Ba-L gp120 proteins. Serum titers

were determined as the highest dilution of immune serum

producing ELISA values (A450 nm) greater than or equal to two

times the binding detected with a corresponding dilution of pre-

immune serum. Protein boost-mediated fold increase in ELISA

titers was calculated as follows: ELISA Titers (DNA+Protein)/

ELISA Titers (DNA). As expected, serum from naı̈ve animals had

no reactivity with Env and Gag antigens in the ELISA (data not

shown).

Murine IFNc ELISPOTThe IFNc ELISPOT assay was performed using murine

splenocytes according to the manufacturer’s protocol (UCyTech,

Netherlands) as previously described [45,46].

Cytometric Bead ArrayCytometric bead array (BD Biosciences, San Diego, CA) was

performed to quantitate secreted Th1 (IFNc, IL-2, TNFa), Th2

(IL-4, IL-5, IL-6) and Th17 (IL-17) cytokines from supernatants of

peptide-stimulated murine splenocytes as described [46,47].

Statistical AnalysisStatistical significance of Nedd4-induced HIV-1 p24 or gp120

levels, in vitro, was demonstrated using a one way ANOVA

followed by Tukey’s multiple comparison test. Statistical signifi-

cance of Nedd4-induced vaccine-specific immune responses,

in vivo, was demonstrated using the Kruskal-Wallis nonparametric

test followed by Dunn’s multiple comparison test.

Results

Increased Nedd4 Protein Levels Following SHIV SF162P3-Infection of Rhesus Macaques

Changes in intracellular Nedd4 protein expression were initially

evaluated post-intra-rectal challenge of rhesus macaques with

SHIVSF162P3. Nedd4 protein levels were assayed, using flow

cytometry, following mucosal challenge of naı̈ve rhesus macaques

using a single, high dose concentration of virus. Following

challenge, plasma viral RNA load was found to increase with

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peak viremia noted at day 14 post-challenge (Figure 1A). Absolute

CD4 T cell counts were quantified at days 0, 14, 28, 42, and 66

post-challenge and were found to decrease in 3 of 4 macaques

post-challenge (Figure 1B). By contrast, macaque L861 showed an

increase in CD4 T cell counts post-challenge. Intracellular staining

of Nedd4 using peripheral blood mononuclear cells was performed

and both histogram representations (Figure 1D) as well as

graphical representations (Figure 1E) are shown for Nedd4

expression in CD4 and CD8 T cells. Mean Fluorescence

Intensities (MFI) of Nedd4+ in CD4+ (Figure 1E, upper panel)

and CD8+ (Figure 1E, lower panel) T cells revealed an increase in

Nedd4 protein levels at day 7 post infection followed by a decline

by day 21 and then an increase by day 42.

Increased Levels of Extracellular and Intracellular HIV-1Gag and Env by Nedd4

Given the increased Nedd4 protein levels noted following SHIV

infection of rhesus macaques, transient transfection assays were

performed to assess a potential role for Nedd4 in viral replication

as determined by HIV-1 p24 Gag and/or gp120/gp160 produc-

tion. Using a radio-immunoprecipitation assay, it was found that

293 cells co-transfected with plasmid DNA expressing Nedd4 and

the HIV-1 molecular clone, HXB2, yielded increased levels of

secreted (Figure 2A) and intracellular p24 Gag compared to

HXB2 alone (Figure 2B). Similarly, Nedd4/HXB2 co-transfection

augmented secreted gp120 (Figure 2A) and intracellular gp160

(Figure 2B) compared to HXB2 alone.

Figure 1. Increased Nedd4 Protein Expression Following SHIVSF162P3 Infection of Rhesus Macaques. Rhesus macaques were challengedintra-rectally with SHIV162P3. Plasma viral RNA load (A) was measured, using Real Time NASBA, at days 7, 10, 14, 21, 28, 35, 42, 52, 70 and 98 post-challenge and CD4 T cell counts (B) were quantified at days 0, 14, 28, 42, and 66 post challenge as outlined in the Materials and Methods (B).Intracellular staining of Nedd4 was performed using macaque peripheral blood mononuclear cells (D, E). Cells were acquired using an LSRII cytometer(BD Biosciences) and histogram representations (D) as well as graphical representations (E) are shown. Mean Fluorescence Intensities (MFI) ofNedd4+CD4+ T cells (E, upper panel) and Nedd4+CD8+ T cells (E, lower panel) are graphically represented for days 0, 7, 21 and 42 post-challenge.doi:10.1371/journal.pone.0091267.g001

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Increased HIV-1 p24 in Cell Supernatants by Nedd4 isDependent on Ca2+/Calmodulin-Regulated PhospholipidBinding C2 Domain and Not Ubiquitin Ligase Activity

In order to identify the protein domains that may be critical for

the Nedd4-mediated increase in p24 levels, 293 cells were co-

transfected with HXB2 and various Nedd4 protein domain

mutants. Nedd4 is comprised of several protein domains including

a Ca2+/calmodulin-regulated phospholipid binding domain (C2),

3–4 WW protein binding domains (WW1–4) and an ubiquitin

ligase enzymatic (HECT) domain (Figures 3A, 4A). Initially, co-

transfection of 293 cells was performed with HXB2 and plasmid

DNA expressing either wild type rat Nedd4 (rNedd4) or a C/S

mutant Nedd4 (rNedd4CSmut) that lacks ubiquitin ligase activity

(Figure 3A). As shown in Figure 3B, transfection with either

HXB2/rNedd4 or HXB2/rNedd4CSmut was found to yield

significantly increased supernatant p24 levels at 48 hr (p,0.01)

compared to levels obtained when cells were transfected with

HXB2 alone. Supernatant p24 levels were not found to be

statistically different (p.0.05) when comparing HXB2/rNedd4

and HXB2/rNedd4CSmut cultures. Western blot analyses dem-

onstrated a low but detectable level of endogenous Nedd4 protein

(Figure 3C). Nedd4 protein bands were markedly enhanced

following rNedd4 and rNedd4CSmut transfection. These findings

suggest that the mechanism by which Nedd4 is mediating an

increase in p24 levels is not dependent on ubiquitin ligase activity.

Transfection studies were next performed using HXB2 co-

expressed with either human Nedd4 (hNedd4), human Nedd4

lacking the C2 domain (hNedd4C2mut) or a specific splice variant

of Nedd4-2 that lacks the C2 domain (Figure 4A). Consistent with

the transfection studies described above, using rNedd4, exogenous

co-expression of HXB2 and hNedd4 was found to significantly

(p,0.01) increase supernatant p24 compared to cells transfected

with HXB2 alone (Figure 4B). By contrast, transfection of 293 cells

with hNedd4C2mut or hNedd4-2 did not result in a statistically

significant (p.0.05) increase of p24 levels compared to HXB2

alone. Taken together, these findings suggest that the increase in

supernatant p24 levels, by Nedd4, is dependent on the Ca2+/

calmodulin regulated phospholipid binding C2 domain and not

ubiquitin ligase activity.

Nedd4 siRNA Reduces Supernatant p24 Levels in HXB2-Transfected Cells

Given that Nedd4/HXB2 co-expression in 293 cells demon-

strated increased p24 levels (Figures 2–3, 4B), we hypothesized

that inhibiting endogenous levels of Nedd4 may result in decreased

levels of secreted p24. Hence, we co-transfected 293 cells with

HXB2 and either a Nedd4-targeted siRNA previously shown to

inhibit Nedd4 expression, or an irrelevant control siRNA targeting

GFP [48]. When cell supernatants from these transfections were

assayed for p24, levels were found to be markedly reduced in

HXB2/siRNA-transfected cells as compared to HXB2-transfected

cultures (Figure 4D). In contrast, p24 levels remained unchanged

in samples where co-transfection included HXB2 and a control

siRNA. While western blot analyses demonstrated that Nedd4-

targeted siRNA could reduce endogenous Nedd4 protein levels, a

more pronounced reduction was evident when cells were co-

transfected with Nedd4 and siRNA compared to Nedd4 alone

(Figure 4E).

Figure 2. Increased Levels of Extracellular and Intracellular HIV-1 Gag and Env Proteins by co-expression of Nedd4. 293 cells weretransiently transfected with HXB2 and a plasmid encoding human Nedd4 (hNedd4) as described (Materials and Methods). At 32 hr post-transfection,a radio-immunoprecipitation assay (RIPA) was performed by labeling cells for 12.5 hr with 35S-Met, immunoprecipitating target proteins using anti-HIV+ human serum, and resolving bands from cell media (A) and lysates (B) using 10% SDS-PAGE and autoradiography at –70uC. Bands correspondingto HIV-1 gp160, gp120, p55 and p24 proteins are shown.doi:10.1371/journal.pone.0091267.g002

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Exogenous Nedd4 Does Not Affect LTR Promoter ActivityWe next tested the possibility that the Nedd4-mediated increase

in p24 levels was driven by an increase in HIV-1 promoter

activity. To this end, Jurkat T cells were transfected with an HIV-1

LTR-luciferase reporter construct in the absence or presence of

hNedd4. At 24 hr post-transfection, cells were either left

unstimulated or treated with phorbol ester [phorbol 12-myris-

tate-13-acetate (PMA)], calcium ionophore [ionomycin (Iono)] or

both PMA and Iono. In all treatment conditions tested, HIV-1

LTR activity remained unchanged by the presence of hNedd4

expression (Figure 5A).

Nedd4 Mediates Increased p24 Levels in Gag-Transfected293 Cells

We next investigated whether Nedd4 could mediate an increase

in extracellular p24 levels when HXB2 was replaced with a

plasmid DNA expressing Gag in co-transfection experiments.

Hence, 293 cells were transfected with a GFP-Gag fusion

construct in the presence and absence of hNedd4 and supernatant

p24 levels were measured as described (Materials and Methods).

Consistent with data shown with HXB2 (Figures 3–4), hNedd4

was found to significantly (p,0.01) increase supernatant p24 levels

when cells were co-transfected with GFP-Gag compared to cells

transfected with GFP-Gag alone (Figure 5B). This was noted both

at 24 hr and 48 hr post-transfection.

De Novo Protein Synthesis is Not Required for Nedd4-Mediated Increase in p24 Levels

In an effort to further understand the mechanism by which

Nedd4 increases HIV-1 p24, we asked whether de novo protein

synthesis is required for this modulation. A preliminary experi-

ment was performed in which 293 cells were transfected with

Figure 3. Ectopic expression of Nedd4 augments supernatant HIV-1 p24 levels Independent of HECT Domain Function. 293 cellswere transfected with HXB2 and a plasmid encoding either rat Nedd4 (rNedd4) or a rat Nedd4 catalytic domain mutant (rNedd4CSmut) and at 48 hrpost-transfection, cell supernatants were collected and p24 assays were performed as described (Material and Methods). Schematics highlighting theCa2+/Calmodulin-regulated phospholipid binding domain (C2), WW protein binding domains (WW1–3) and E3 catalytic domain (HECT) for rNedd4and rNedd4CSmut are shown (A). Mean p246 standard error values, calculated from data obtained from transfections conducted with rNedd4 (n = 7)and rNedd4CSmut (n = 5), are graphically represented (B). Statistical significance was shown for the increased p24 levels by rNedd4 (rNedd4 vs controlat 48 hr: p,0.01; rNedd4CSmut vs control at 48 hr; p,0.01) using a one way ANOVA followed by Tukey’s multiple comparison test. No statisticaldifference was shown for rNedd4CSmut compared to rNedd4 at 48 hr (p.0.05) using this analysis. Immunoblot analysis, using a rabbit anti-mouseNedd4 antibody, was performed on detergent soluble cell fractions as described (Materials and Methods). Nedd4 and Nedd4CSmut protein bands areindicated (arrows) (C).doi:10.1371/journal.pone.0091267.g003

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Figure 4. Increased p24 induced by Nedd4 Mediated by C2 domain. 293 cells were transiently transfected with HXB2 and a plasmidencoding human Nedd4 (hNedd4), human Nedd4 C2 deletion mutant (hNedd4C2mut) or a human Nedd4-2 splice variant that lacks the C2 domain(hNedd4-2) as described (Materials and Methods). Cell supernatants were collected at 48 hr post-transfection and p24 assays were performed. Aschematic highlighting the Ca2+/Calmodulin-regulated phospholipid binding domain (C2), WW protein binding domains (WW1–4) and E3 catalyticdomain (HECT) for hNedd4 is shown (A). Mean p246 standard error values were calculated from data obtained from transfections conducted withhNedd4 (n = 3), hNedd4C2mut (n = 3) and hNedd4-2 (n = 4) and are graphically represented (B). Statistical significance was shown for the increasedp24 levels by hNedd4 (hNedd4 vs control at 48 hr: p,0.01) using a one way ANOVA followed by Tukey’s multiple comparison test. Statisticalsignificance was not seen for hNedd4C2mut or hNedd4-2 (hNedd4C2mut or hNedd4-2 vs control at 48 hr: p.0.05). Western blot analyses, using arabbit anti-mouse Nedd4 antibody, was performed to measure Nedd4, Nedd-2 and Nedd4-C2mut protein levels post-transfection as described(Materials and Methods). Nedd4 (dotted arrow), Nedd-2 (arrow), Nedd4-C2mut (arrow head) and actin protein bands are indicated (C). For siRNAexperiments, 293 cells were transiently transfected with HXB2 alone or HXB2 with either Nedd4-targeted siRNA or irrelevant control siRNA (Materialsand Methods). Following 24 hrs, supernatant p24 levels were measured and mean 6 standard error values are graphically represented (D). Westernblot analysis of Nedd4 protein levels in 293 cells transfected with HXB2, HXB2/Nedd46 siRNA is shown (E). Statistical significance, using a one wayANOVA followed by Tukey’s multiple comparison test, was shown for the decreased supernatant p24 levels from cells co-transfected with HXB2/siRNA (p,0.05) but not HXB2/control siRNA (p.0.05) as compared to cells with HXB2 alone.doi:10.1371/journal.pone.0091267.g004

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GFP-Gag in the absence or presence of hNedd4 for 24 hours

followed by treatment with protein synthesis inhibitor, cyclohex-

imide. Levels of Gag protein were then measured, by western blot

analysis, at 0, 6, 12 and 25 hr post-cycloheximide treatment

(Figure 5C). While Gag protein levels were found to decline over

time following cycloheximide addition in the absence of ectopic

hNedd4 expression, Gag remained elevated and did not decline in

hNedd4-transfected samples (Figure 5C). Our data supports that

idea that Nedd4 does not require de novo protein synthsis to

mediate increased levels of HIV-1 p24.

Nedd4 Co-Adminstration of BALB/c mice YieldsDifferential Vaccine-Specific Humoral and CellularImmune Responses

Given the ability of Nedd4 to augment both intracellular and

secreted HIV-1 p24 and gp120, we hypothesized that such

increases could lead to augmented humoral and cellular immune

responses following co-injection of plasmid DNA expressing

Nedd4 and HIV antigens to BALB/c mice. Prior to immunizing

mice, 293 cells were transiently transfected with plasmid DNA

expressing HIV-1 gag or env and increasing concentrations of

hNedd4. At 48 hours post-transfection, the levels of p24 and

gp120 were assayed, by ELISA, using lysed cell pellets and cell

supernatants (Figure 6A, Figure 6B). A Nedd4 dose-dependent

increase in supernatant p24, intracellular p24, supernatant gp120

and intracellular gp120/gp160 was noted in these transfections.

BALB/c mice (n = 5 per group; 5–6 week old females) were then

immunized intramuscularly at weeks 0, 2 and 4 with plasmid DNA

expressing hNedd4 alone (Group 1), HIV-1 gag+env (Group 2) or

hNedd4+HIV-1 gag+env (Group 3) as described (Materials and

Methods). Group 4 mice (n = 3) were left un-immunized and

served as naı̈ve controls. Mice were sacrificed at week 6 to evaluate

serum antibody responses and splenocyte T-cell responses. Nedd4

co-administration was found to increase vaccine-induced anti-p24

(Figure 7A; p,0.05). While there was a trend of Nedd4-mediated

increases in anti-gp120 (Figure 7B; p.0.05), this observation was

not found to be statistically significant. In order to test the

hypothesis that hNedd4 co-administration could increase Gag-

and/or Env-specific Th1/Th2/Th17 responses, we assessed

peptide-specific IFNc, TNFa, IL-2, IL-4, IL-6 and IL-17 cytokine

levels following in vitro peptide stimulation of splenocytes. Gag

(consensus A, consensus B, consensus C) and Env peptide-specific

IFNc production, quantified by ELISPOT assay, was not found to

be significantly modulated by hNedd4 co-administration

(Figure 8A, Figure 8C). Nedd4 also did not significantly alter the

levels of Gag- or Env-specific IL-2 and IL-4, as measured by CBA

(data not shown). Although not statistically significant (p = 0.6905–

0.8413), a trend of increased Gag-specific TNFa (Figure 8B, left

panel), IL-6 (Figure 8B, middle panel), and IL-17A (Figure 8B,

right panel) was noted with hNedd4 co-administration. This trend,

however, was not noted for Env-peptide specific cytokine

responses in T-cells (Figure 8D).

Discussion

It has now been demonstrated by a number of laboratories that

HIV-1 exploits intracellular molecules to facilitate its trafficking

through and budding from infected host cells [2,5,49–51]. Not

surprising, it has also been shown that HIV-1 does not simply rely

upon one molecule such as Tsg101 to bud, but it appears that the

virus has evolved to make use of several intracellular proteins

including AIP1/ALIX [1]. Given that HIV-1 Gag is post-

translationally ubiquitinated and that a role for Nedd4 in HIV-1

trafficking and budding has not been fully explored, we

Figure 5. Nedd4 expression stabilizes intracellular HIV-1protein levels but does not increase LTR promoter activity.Jurkat T cells were transfected with LTR-luciferase and pRL-TK-renillareporter constructs in the absence or presence of hNedd4 (A). At 24 hrpost-transfection, cells were either left unstimulated or stimulated withPMA, ionomycin (Iono) or with PMA+Iono. Jurkat T cells weretransfected with LTR-luciferase and pRL-TK-renilla reporter constructsin the absence or presence of hNedd4. At 24 hr post-transfection, cellswere either left unstimulated or stimulated with PMA, ionomycin (Iono)or with PMA+Iono. A dual luciferase assay was performed as per themanufacturer’s protocol (Materials and Methods) and mean 6 standarderror values for n = 3 experiments are shown graphically (A). Nostatistical significance, using a one way ANOVA followed by Tukey’smultiple comparison test, was shown for the supernatant LTR activityfrom cells co-transfected in the absence or presence of hNedd4 (p.0.05). Alternatively, 293 cells were transiently transfected with a GFP-Gag fusion construct in the absence or presence of hNedd4 (B). At 24 hrand 48 hr post-transfection, p24 assays were performed as described(Experimental Procedures). Mean p246 standard error values (n = 2)were calculated, graphically represented and statistical significance wasshown for GFP-Gag/hNedd4 versus GFP-Gag (at 24 hr, 48 hr: p,0.05).293 cells were transiently transfected with a GFP-Gag fusion constructin the absence or presence of hNedd4 were treated with cycloheximideat 24 hr post-transfection. Immunoprecipitations were performed usinga rabbit anti-GFP polyclonal antibody followed by immunoblottingusing a mouse anti-GFP monoclonal antibody on detergent-soluble cellfractions at 0, 6, 12, and 25 hr post-cycloheximide treatment (C).doi:10.1371/journal.pone.0091267.g005

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Figure 6. Increased Levels of Extracellular and Intracellular p24 Gag and gp120 Env Proteins by Nedd4. 293 cells were transientlytransfected with a plasmid DNA expressing a clade B Mosaic Gag (A) or HIV-1BaL gp120 (B) and increasing concentrations of hNedd4 as described(Materials and Methods). At 48 hr post-transfection, cell supernatants or cell lysates were assayed for p24 (A) or gp120 (B) by ELISA. Levels (pg/ml) ofp24 (A) and gp120 (B) are represented graphically for supernatants (blue line) or cell lysates (red line).doi:10.1371/journal.pone.0091267.g006

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investigated a potential role for this E3 ubiquitin ligase in HIV

infection and pathogenesis. We hypothesized that if Nedd4 plays a

key role in HIV pathogenesis, induction of Nedd4 would be

observed post-infection of rhesus macaques with Simian-Human

Immunodeficiency Virus and/or Simian Immunodeficiency Virus.

Consistent with a role for Nedd4 in HIV infection, intra-rectal

challenge of rhesus macaques with SHIVSF162P3 demonstrated an

increase in Nedd4 protein post-infection (Figure 1). It is possible

that HIV may be exploiting E3 ligases to facilitate virus replication

and/or budding. Alternatively, we cannot rule out the possibility

that early induction of Nedd4 and other E3 ligases represents, in-

part, a host antiviral response that is exploited by the virus to

mediate increased replication, budding and infection. This idea is

consistent with the noted increase in Nedd4, by day 7 post

Figure 7. Differential Effects on Vaccine-Specific Humoral Immune Responses by Nedd4 Co-Adminstration of BALB/c mice.Intramuscular immunization of BALB/c mice (n = 5 per group; 5–6 week old females) was performed at weeks 0, 2 and 4 with plasmid DNA expressinghuman Nedd4 alone (Group 1), HIV-1 gag +env (Group 2) or human Nedd4+ HIV-1 gag+env (Group 3) as described (Materials and Methods). Group 4mice (n = 3) were left un-immunized and served as naı̈ve controls. Mice were sacrificed at week 6 to evaluate serum antibody responses (A, B).Vaccine-induced anti-p24 (A) and anti-gp120 (B) antibody levels were quantitated and reciprocal mean ELISA titers 6 standard errors are graphicallydepicted. Statistical significance, using the Kruskal-Wallis nonparametric test followed by Dunn’s multiple comparison test, was noted in anti-p24titers (Gag+Env+Nedd4 vs Gag+Env; P,0.05) but not anti-pg120 titers (Gag+Env vs Gag+Env+Nedd4: p.0.05).doi:10.1371/journal.pone.0091267.g007

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infection (Figure 1E) that precedes peak viremia noted at day 14

(Figure 1A). We speculate that the subsequent decline in Nedd4

protein levels, by day 21, may reflect induction of a negative

signaling pathway or cell death. A second round of increased

Nedd4 levels, by day 42, may signal another host-antiviral

response. Ongoing studies seek to understand the pathways

involved in such Nedd4 modulation and if other E3 ligases are

induced post infection with HIV/SIV/SHIV.

While Nedd4 was shown to augment intracellular and

supernatant p24 levels in HXB2-transfected 293 cells, the increase

was not dependent on ubiquitin ligase activity but rather on the

Ca2+/calmodulin regulated phospholipid binding C2 domain

(Figures 2–4). We therefore speculated that Nedd4 may be either

functioning to increase viral expression, via augmented long

terminal repeat (LTR) activity, or serving as an adapter to stabilize

p24 protein levels and facilitate HIV-1 egress. While ectopic

expression of Nedd4 did not affect LTR activity, (Figure 5A)

transfection studies using Nedd4/Gag in conjunction with the

protein synthesis inhibitor, cycloheximide, revealed a sustained

level of Gag post-cycloheximide treatment that was not seen in the

absence of Nedd4 (Figure 5C). Such findings are consistent with

Nedd4 playing a role in stabilizing HIV-1 p24 and facilitating viral

budding. More studies are needed, however, to assess the ability of

Nedd4 to interact with and stabilize other HIV-1 proteins

including gp160 or gp120. Previous work has shown that

uncleaved gp160 is processed through lysosomes and only a small

percentage of gp160 is cleaved to form the mature gp120 [52].

Hence, it is possible that Nedd4 could also play a role in the

processing of the HIV envelope.

Several laboratories have now clearly demonstrated that Tsg101

binds HIV-1 Gag and thereby plays a critical role in mediating

HIV-1 viral budding through late endosomes [49,50]. We

therefore speculated that if Nedd4 functions as an adaptor in

facilitating HIV-1 trafficking and egress, it may do so by binding

Tsg101 and thus recruiting the Tsg101/Gag complex to late

endosomes. Consistent with this hypothesis, preliminary co-

immunoprecipitation studies found an association of Tsg101 and

Nedd4 (data not shown). While additional studies are needed to

confirm this association, our findings suggest that HIV exploits

Tsg101 and Nedd4 for trafficking and egress from target cells.

HIV-1 has been shown to use different proteins to route the virus

through different budding compartments [50,53]. Studies to

evaluate these pathways have demonstrated that HIV-1 budding

can occur in primary macrophages and monocytic cell lines

principally via late endosomes/MVBs [54,55] and in T cells via

the plasma membrane [53]. In our laboratories, Nedd4-mediated

increased p24 levels were found to be similar in both T cell (H9,

CEM) and monocytic (THP-1, U937) cell lines tested (data not

shown). Our data suggests that Nedd4-mediated egress can occur

via either late endosomes/MVBs or the plasma membrane.

Given that Nedd4 was shown to enhance intracellular and

extracellular p24 Gag and gp120 Env, in co-transfection studies

using plasmid DNA expressing either viral protein (Figure 6), we

reasoned that Nedd4 co-administration to mice in the context of

HIV vaccination could enhance vaccine-specific antibody and T-

cell responses. Consistent with this hypothesis, Nedd4 co-injection

in BALB/c mice was indeed able to augment anti-p24 antibodies

(Figure 7A). Given the weak levels of anti-Gag antibodies versus

robust levels of anti-Env antibodies (Figure 7B) noted following

Figure 8. Effects of Nedd4 Co-Adminstration on Vaccine-Specific Cellular Immune Responses in BALB/c mice. Intramuscularimmunization of BALB/c mice (n = 5 per group; 5–6 week old females) was performed at weeks 0, 2 and 4 with plasmid DNA expressing human Nedd4alone (Group 1), HIV-1 gag +env (Group 2) or human Nedd4+ HIV-1 gag+env (Group 3) as described (Materials and Methods). Group 4 mice (n = 3)were left un-immunized and served as naı̈ve controls. Mice were sacrificed at week 6 to evaluate splenocyte T-cell responses. Gag (A) and Env (C)peptide (consensus A, consensus B, consensus C)-specific IFNc production was quantified by ELISPOT assay and mean spots per million splenocytevalues 6 standard errors are graphically represented. Gag peptide-specific TNFa (B, left panel), IL-6 (B, middle panel), IL-17A (B, right panel) and Envpeptide specific TNFa (D, left panel), IL-6 (D, middle panel), IL-17A (D, right panel) cytokines were quantified by CBA from the supernatants of peptidestimulated splenocytes and are represented graphically as mean pg/ml values 6 standard error. Statistical significance was assessed using theKruskal-Wallis nonparametric test followed by Dunn’s multiple comparison test. No statistical difference in IFNc, TNFa, IL-6 and IL-17A and valueswere found (Gag+Env vs Gag+Env+Nedd4: p.0.05).doi:10.1371/journal.pone.0091267.g008

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three DNA administrations of antigen alone, it is not surprising

that a Nedd4-induced increase in humoral responses was only

evident for anti-p24. Future studies will seek to lower the dose of

plasmid DNA expressing env in an effort to assess if Nedd4 can

mediate a dose sparing effect. While Nedd4 co-administration did

not affect Gag- or Env-specific IFNc T-cell responses (Figure 8A,

Figure 8C), a trend of increased Gag-specific IL-6, IL17A and

TNFa was noted (Figure 8B). Interestingly, the E3 ligase adaptor,

Ndfip1, has been shown to regulate Th17 differentiation in mice

[56]. Whether this increase in IL-6 and IL17A is a general

function of E3 ligases or whether it is a Nedd4-specific phenomena

remains to be determined. Given that the Nedd4-mediated

increase in HIV-1 p24 was dependent on the C2 domain, future

studies will focus on evaluating if increased humoral and cellular

responses are noted in mice with co-administration of a plasmid

DNA expressing the minimal C2 domain rather than the full

Nedd4 sequence. Given that our findings showed differential

effects, by Nedd4, on antibody and T-cell responses, more studies

are needed to understand if such differences are due, in-part, to

the use of separate plasmids for antigens and Nedd4 expression.

Future studies will use a bi-cistronic plasmid to ensure that both

antigens and Nedd4 are delivered to and expressed in the same

cells. Overall, our findings that Nedd4 increases the levels of HIV

proteins warrants further investigation into possible mechanisms of

action as well as possible applications in the context of HIV

vaccination.

Acknowledgments

The authors thank Dr. Marvin Reitz (Institute of Human Virology,

University of Maryland) for the generous gift of HXB2, Dr. Hughes Abriel

(Institute of Pharmacology and Toxicology, University of Lausanne,

Lausanne, Switzerland) for the generous gifts of hNedd4 and hNedd4-2

constructs and Dr. Daniela Rotin (Hospital for Sick Children, University of

Toronto) for the rNedd4 and rNedd4CSmut constructs. We would also

liked to thank Jim Treece and Sharon Orndorff for their assistance in

conducting the murine immunogenicity study.

Author Contributions

Conceived and designed the experiments: BL MCH ADC. Performed the

experiments: BL SW LH LG MCH. Analyzed the data: BL MCH ADC.

Wrote the paper: ADC.

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